The acid resistance
assay of Castanie-Cornet et al. (1999), as modified by David Graham (Giles & Graham, 2007), was utilized. Acid resistance was expressed as the percentage of viable bacteria remaining after one-hour acid shock compared to the number of viable bacteria determined immediately following acid shock. The highly conserved chlamydial AaxB peptide 137HAKMWLKKSLQHELDLRS154 (part of the α subunit) was commercially synthesized by Pierce Custom Antibody Production Service and used to raise polyclonal rabbit antibodies using the standard 90-day protocol. Escherichia coli Rosetta-gami2 (DE3) was transformed with pET-19b carrying aaxB from C. caviae and grown in LB containing 100 μg mL−1 ampicillin to an OD600 nm of 0.6. AaxB expression was induced with 1 mM IPTG for 23 h at 20 °C, and bacteria were collected by centrifugation. Bacteria were resuspended in equilibration Cabozantinib nmr buffer (50 mM monobasic sodium phosphate, 300 mM sodium chloride, and 10 mM imidazole, pH adjusted to 7.4) with 1× protease inhibitor (Roche) and 1× phosphatase inhibitors 2 and 3 (Sigma). Bacteria were lysed via sonication, centrifuged to remove
debris, and the supernatant passed through a 0.45-μm Doxorubicin supplier filter (Millipore). HisPur™ cobalt resin (Thermo Scientific) was applied to the supernatant, and the batch method of purification was carried out as per manufacturer’s instructions. Purified protein samples were eluted (50 mM sodium phosphate, 300 mM sodium chloride,
500 mM imidazole, pH adjusted to 7.4) and then applied to a 3K Amicon filter (Millipore) for concentration. Samples were resuspended in 50 mM Bis–Tris buffer (pH 6.0) and quantified by the Bio-Rad Protein Assay (Bio-Rad). Protein identity and purity were assessed using SDS-PAGE followed by Coomassie Brilliant Blue Staining or Western blotting with the anti-AaxB antibody (at a 1:250 dilution). Chlamydia were grown in and harvested from mouse fibroblast L2 cells. EBs were titered using an infection-forming unit assay (IFU) and stored at -80 °C in sucrose–phosphate–glutamic acid buffer (SPG; 7.5% w/v sucrose, 17 mM Na2HPO4, 3 mM NaH2PO4, 5 mM l-glutamic acid, pH 7.4) until use (Binet et al., 2010). For not time course experiments, L2 cells were infected at a MOI of 5 (10-h samples) and MOI of 1 (20-, 30-, and 44-h samples), or mock-infected (Giles et al., 2009). Samples were disrupted directly in Laemmli buffer and run on 12% SDS-PAGE gels for Western blot analysis with either anti-AaxB antibodies or anti-Hsp60 antibodies (provided by Dan Rockey, Oregon State University; Yuan et al., 1992). Detection of Hsp60 (60 kDa heat shock protein, GroEL) was used to demonstrate successful infection and equal loading of protein. To detect AaxB in EBs, bacteria were disrupted in Laemmli buffer, and 1 × 107 IFU was used per SDS-PAGE gel lane. The AaxB sequences from the available Chlamydia genome projects were aligned to assess amino acid variability.